Upregulation of the TWEAK/Fn14 pathway is involved in vitreous neovascularization in a mouse model of ROP

Abstract

Purpose: Retinopathy of prematurity (ROP) is a potentially blinding disease that affects preterm babies. ROP is thought to occur due to relative hyperoxia of the extra-uterine environment. This results in constriction and obliteration of the immature vessels, leading to retinal ischemia and vitreo-retinal neovascularization. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, have been implicated in angiogenesis but their role in retinal diseases is unknown. In this study, we tested the hypothesis that the TWEAK/Fn14 pathway is activated and involved in vitreo-retinal neovascularization.

Methods: ROP was induced by maintaining C57BL/6J mice in 75 % oxygen from postnatal day (P) 7 to P12, followed by a return to room air from P12 to P17. Hyperoxia treatment was initiated on postnatal day (P)14. Primary human retinal microvascular endothelial cells (HRMECs) were transfected with HIF-1apha or treated with TWEAK. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to expression of Fn14 and TWEAK.

Results: Fn14 mRNA was increased by 2.3-4.5 folds from P13 to P17 in ROP retinas, whereas TWEAK level was slightly decreased. These alterations were normalized by hyperoxia treatment and were more striking in isolated retinal vessels. Fn14 protein was highly expressed in neovascular tufts in ROP rather than normal vessels in room air control retinas. There was a discernible shift in the immunoreactivity of Fn14 and TWEAK from the neuronal layers in the normal retina to the neovascular tufts in that of ROP. Blockade of TWEAK/Fn14 significantly prevented retinal neovascularization while slightly accelerated revascularization, without affecting VEGF expression. In contrast, activation of Fn14 positively regulated survival pathways in the Bcl2 family and robustly enhanced HRMEC survival. Furthermore, gene analysis revealed the regulatory region of Fn14 gene contains several conserved hypoxia inducible factor (HIF)-1alpha binding sites. Overexpression of HIF-1alpha prominently increased levels of Fn14 mRNA and protein in HRMECs. This was further boosted by VEGF treatment.

Conclusions: The TWEAK/Fn14 pathway is involved in the development of pathological retinal neovascularization during ROP. HIF-1alpha and VEGF are implicated in the upregulation of Fn14.

Keywords: 609 neovascularization • 706 retinopathy of prematurity • 490 cytokines/chemokines